Methods and Systems for Purifying Non-Complexed Botulinum Neurotoxin

ABSTRACT

Methods and systems for chromatographically purifying a  botulinum  neurotoxin are provided. These methods and systems allow for efficient purification of a non-complexed form of the  botulinum  neurotoxin in high purity and yield that can be used as an active ingredient in pharmaceutical preparations.

This application claims the benefit of priority to U.S. ProvisionalApplication No. 61/253,810, filed on Oct. 21, 2009. The contents of thisU.S. Provisional Application are hereby incorporated by reference intheir entirety.

FIELD OF THE INVENTION

This invention relates generally to chromatographic methods and systemsfor purifying free botulinum neurotoxin from cell cultures to produce ahigh purity, high potency product.

BACKGROUND OF THE INVENTION

Botulinum toxin is a neurotoxic protein produced by the bacteriumClostridium botulinum, as well as other Clostridial species, such asClostridium butyricum, and Clostridium baraffi. The toxin blocksneuromuscular transmission and causes a neuro-paralytic illness inhumans and animals, known as botulism. C. botulinum and its sporescommonly occur in soil and putrefying animal carcasses, and can grow inimproperly sterilized or improperly sealed food containers, which arethe cause of many botulism cases. Botulism symptoms can includedifficulty walking, swallowing, and speaking, and can progress toparalysis of the respiratory muscles and finally death.

Botulinum toxin type A is the most lethal natural substance known toman. In addition to serotype A, six other generally immunologicallydistinct botulinum toxins have been characterized, namely botulinumtoxin serotypes B, C₁, D, E, F, and G. The different serotypes can bedistinguished by neutralization with type-specific antibodies and varyin severity of paralysis they evoke and the animal species they mostlyaffect. The molecular weight of the botulinum toxin protein molecule,for each of the known botulinum toxin serotypes, is about 150 kD,composed of an about 100 kD heavy chain joined to an about 50 kD lightchain. Nonetheless, the botulinum toxins are released by Clostridialbacteria as complexes of the 150 kD toxin with one or more non-toxinproteins. For example, botulinum toxin type A exists as 900 kD, 500 kDand 300 kD complexes (approximate molecular weights).

Despite the known toxic effects, Botulinum toxin type A is clinicallyused to treat a variety of indications, including, e.g., neuromusculardisorders characterized by skeletal muscle hyperactivity. For example,BOTOX® is the trademark of a botulinum toxin type A complex availablecommercially from Allergan, Inc., of Irvine, Calif. Botulinum toxin typeA finds use, for example, in the treatment of essential blepharospasm,strabismus, cervical dystonia, and glabellar line (facial) wrinklesOther serotypes also have been used clinically. A botulinum toxin typeB, for example, has been indicated for use in treating cervicaldystonia. The botulinum toxins are believed to bind with high affinityto the presynaptic membrane of motor neurons, translocate into theneuron, and thereafter block the presynaptic release of acetylcholine.

The botulinum toxin for clinical use is typically isolated from cellculture and various purification approaches have been used.Historically, the toxin is purified in complexed form by a series ofprecipitation and tangential flow filtration steps. See, e.g., SchantzE. J., et al., Properties and use of botulinum toxin and other microbialneurotoxins in medicine, Microbiol Rev 1992 March 56(1):80-99. Suchapproaches have provided relatively low yields, however, typically lessthan about 10%. Other approaches have used size exclusion, ion exchange,and/or affinity chromatography. See, e.g., Schmidt J. J., et al.,Purification of type E botulinum neurotoxin by high-performance ionexchange chromatography, Anal. Biochem. 1986 July; 156(1):213-219;Simpson L. L., et al., Isolation and characterization of the botulinumneurotoxins, Harsman S, ed. Methods in Enzymology. Vol. 165, MicrobialToxins: Tools in Enzymology San Diego, Calif.: Academic Press; vol 165:pages 76-85 (1988); Kannan K., et al., Methods development for thebiochemical assessment of Neurobloc (botulinum toxin type B), Mov Disord2000; 15 (Suppl 2):20 (2000); Wang Y. C., The preparation and quality ofbotulinum toxin type A for injection (BTXA) and its clinical use,Dermatol Las Faci Cosm Surg 2002; 58 (2002); and U.S. Pat. Appl. Publ.No. 2003/0008367.

Still other approaches have focused on just one of the toxin's heavy orlight chains, rather than a complete and biologically active botulinumtoxin protein. For example, one of the chains is individuallysynthesized by recombinant means. See, e.g., Zhou L., et al., Expressionand purification of the light chain of botulinum neurotoxin A: A singlemutation abolishes its cleavage of SNAP-25 and neurotoxicity afterreconstitution with the heavy chain, Biochemistry 1995; 34(46):15175-81(1995); and Johnson S. K., et al., Scale-up of the fermentation andpurification of the recombination heavy chain fragment C of botulinumneurotoxin serotype F, expressed in Pichia pastoris, Protein Expr andPurif 2003; 32:1-9 (2003). These approaches, however, require extrasteps to reform a complete and biologically active botulinum toxinprotein.

A more recent approach involves the use of hydrophobic interactionchromatography, mixed mode, and/or ion exchange chromatography to purifya botulinum toxin as a complex. See, e.g., U.S. Pat. Nos. 7,452,697 and7,354,740, which are hereby incorporated by reference.

Accordingly, there is a need in the art for improved purificationmethods for isolating complete botulinum toxin proteins in stable,biologically active, but non-complexed forms. It is therefore an objectof the invention to provide compositions and methods addressing theseand other needs.

The foregoing discussion is presented solely to provide a betterunderstanding of the nature of the problems confronting the art andshould not be construed in any way as an admission as to prior art norshould the citation of any reference herein be construed as an admissionthat such reference constitutes “prior art” to the instant application.

SUMMARY OF THE INVENTION

This invention relates to systems and methods for purifying anon-complexed botulinum toxin. In one embodiment, the method comprisespurifying crude non-complexed botulinum toxin to obtain a purifiednon-complexed botulinum toxin. In this embodiment, the method comprisesloading an anion exchange column with the crude non-complexed botulinumtoxin to capture the non-complexed botulinum toxin on the anion exchangecolumn; eluting the non-complexed botulinum toxin with buffer to give aneluent comprising the non-complexed botulinum toxin; loading a cationexchange column with the eluent from the anion exchange to column topermit capture of the non-complexed botulinum toxin; and eluting thenon-complexed botulinum toxin with another buffer to give an eluent,thereby obtaining a purified non-complexed botulinum toxin.

In certain embodiments, the botulinum toxin complex is itself obtainedby a number of chromatography steps. In some embodiments, a method forobtaining the botulinum toxin complex comprises obtaining a samplecomprising a botulinum toxin complex; loading a hydrophobic interactioncolumn with the sample to permit capture of the toxin, wherein thecaptured botulinum toxin comprises a complexed botulinum toxin; andeluting the complexed botulinum toxin. The non-complexed botulinum toxinis then dissociated from the complex and the non-complexed botulinumtoxin is purified according to the method described above. In someembodiments, the sample is obtained by subjecting a fermentation culturecomprising botulinum toxin to acid to obtain an acid precipitate, whichmay be subjected to additional pre-chromatography purification steps,non-limiting examples of which include tangential flow filtration toconcentrate the insoluble material of the precipitate, nuclease digest,clarifying centrifugation and/or filtration.

In some embodiments, the sample is subjected to a nuclease digestionbefore loading on the hydrophobic interaction column. Preferably, thenuclease is derived in an animal-product-free process, and even morepreferably the entire purification process is animal product free or atleast substantially animal product free.

In some embodiments, the sample to be used in the chromatographicseparations is preferably a supernatant or filtrate fraction.

The purified non-complexed botulinum toxin comprises at least one ofbotulinum toxin type A, B, C₁, D, F, F and G, and preferably a botulinumtoxin type A having a molecular weight of about 150 kD. In somepreferred embodiments, the purified non-complexed botulinum toxin is atleast 98% pure; and/or has an activity of at least 200 LD₅₀ units/ng. Insome embodiments, the method produces a yield of at least about 2 mg/Lfermentation culture. In other embodiments, the method produces a yieldof about 1 to about 2 mg/L fermentation culture.

These and other aspects of the invention will be better understood byreference to the following detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a summary flow chart comparing one embodiment of a processaccording to the instant invention for directly purifying anon-complexed botulinum toxin (FIG. 1A) with a process for purifying acomplexed botulinum toxin (FIG. 1B).

DETAILED DESCRIPTION

This invention relates to systems and methods for purifying anon-complexed botulinum toxin. In certain embodiments, the methodcomprises purifying a crude non-complexed botulinum toxin by loading ananion exchange column with the crude non-complexed botulinum toxin topermit capture of the non-complexed botulinum toxin by the anionexchange column. Non-complexed botulinum toxin is then eluted withbuffer to give an eluent comprising the non-complexed botulinum toxin.The eluent from the anion column is loaded on a cation exchange columnto permit capture of the non-complexed botulinum toxin and the purifiednon-complexed botulinum toxin is eluted with buffer, thereby obtaining apurified non-complexed botulinum toxin.

In some embodiments, the invention provides for the purification ofnon-complexed botulinum toxin in a relatively small number of steps toproduce a high yield, high purity, and high potency product. Processesand systems within the scope of the invention can be used to efficientlyproduce a stable but non-complexed botulinum toxin from fermentationcultures. In other embodiments, the method further comprises providing asample comprising botulinum toxin complex and loading a hydrophobicinteraction column with the sample so as to permit capture of thebotulinum toxin complex by the hydrophobic interaction column. Thebotulinum toxin complex is then eluted from the column with buffer.Crude non-complexed botulinum toxin is dissociated from the botulinumtoxin complex to obtain a mixture comprising the crude non-complexedbotulinum toxin. In this embodiment, the mixture comprising crudenon-complexed botulinum toxin is purified to obtain pure orsubstantially pure botulinum toxin according to the method describedabove.

One aspect of this invention is the recognition that a pharmaceuticalcomposition comprising non-complexed botulinum toxin as an activeingredient can provide greater purity compared to one comprising acomplexed form. Non-toxin proteins typically associated with a botulinumtoxin complex can account for about 90% by weight of the complex. Thus,providing a botulinum toxin as a complex necessarily includes at leastabout 90% by weight of impurities. In other words, at least about 80 toabout 90% by weight of the pharmaceutical composition will includecell-derived impurities that are not part of the active molecule nornecessary for its biological activity. Such impurities, however,represent cell-derived materials that when administered to a patient mayincrease the risk of unwanted immunological reactions to the drug; mayincrease the risk of unwanted side effects; and/or may increase the riskof transmission of pathogenic agents. In contrast, the high purity of anon-complexed product, obtainable by methods and systems describedherein, reduces the amount of host cell impurities that may remain inthe pharmaceutical composition, thereby reducing the attendant risks ofunwanted reactions and/or transmission. Accordingly, processes andsystems described herein can provide a botulinum toxin in a form morereadily suited to the preparation of safer, purer pharmaceuticalcompositions.

Moreover, unlike complexed forms, free botulinum toxin prepared inaccordance with the method described herein does not need to bestabilized for storage in blood-derived products. Botulinum toxin type Acomplex, for example, is typically stabilized in an excipient comprisingalbumin, which is derived from human blood. For example, BOTOX® consistsof a purified botulinum toxin type A complex, human serum albumin, andsodium chloride packaged in vacuum-dried form. The same is true forDysport and Xeomin. While screenings reduce likelihood of contaminationwith pathogenic agents, use of human blood in pharmaceuticalpreparations generally increases the risk of unwanted transmission ofcertain pathogenic agents, e.g., agents which are not or cannot yet bescreened out. In contrast, free botulinum toxin prepared according tothe instant invention can be stably stored, as taught herein, inammonium sulfate. Further, in some preferred embodiments, methods andsystems of the instant invention are substantially, essentially, orentirely animal product free, as discussed herein. The ability to alsostably store the toxin product substantially, essentially, or entirelyanimal-product free, further reduces potential risks associated withanimal-derived products. Accordingly, processes and systems describedherein provide a botulinum toxin in a form particularly suited topharmaceutical applications in terms of safety, e.g., where thepharmaceutical composition may be prepared and stored substantially,essentially, or entirely animal-product free.

In certain preferred embodiments, the processes and systems describedherein are scalable and/or cGMP compliant. Accordingly, methods andsystems described herein may be used on a commercial, industrial scale,to produce non-complexed botulinum toxin for use, e.g., inpharmaceutical compositions. A cGMP compliant process or system refersto one that can comply with the regulatory requirements for current goodmanufacturing practices, as required by the U.S. Code of FederalRegulations. In some preferred embodiments, the non-complexed botulinumtoxin product is particularly suited to large scale production due toits ease of storage and usability, high activity, high purity,stability, and/or improved safety.

“Botulinum toxin” as used herein refers to a neurotoxin protein moleculethat can be produced by a Clostridial bacterium, as well asrecombinantly produced forms thereof. A recombinant botulinum toxin canhave the light chain and/or heavy chain of the toxin protein synthesizedvia recombinant techniques, e.g., by a recombinant Clostridial and/ornon-Clostridial species. “Botulinum toxin” is used interchangeablyherein with the related expressions “botulinum neurotoxin,” “neurotoxin”or simply “toxin.” “Botulinum toxin” encompasses any of the botulinumtoxin serotypes A, B, C₁, D, E, F and G, and also encompasses bothcomplexed and non-complexed forms.

By “complexed form” is meant a botulinum toxin complex comprising abotulinum toxin protein (i.e., the toxin molecule with a molecularweight of about 150 kD) as well as at least one associated nativenon-toxin protein. Non-toxin proteins that make up the complexestypically include non-toxin hemagglutinin protein and non-toxinnon-hemagglutinin protein. Thus complexed forms may comprise a botulinumtoxin molecule (the neurotoxic component) and one or more non-toxinhemagglutinin proteins and/or one or more non-toxin non-hemagglutininproteins. In certain embodiments, the molecular weight of the complex isgreater than about 150 kD. For example, complexed forms of the botulinumtoxin type A can have molecular weights of about 900 kD, about 500 kD orabout 300 kD. Complexed forms of botulinum toxin types B and C₁ can havea molecular weight of 500 kD. Complexed forms of botulinum toxin type Dcan have a molecular weight of about 300 kD or about 500 kD. Finally,complexed forms of botulinum toxin types E and F can have a molecularweight of about 300 kD.

“Non-complexed” botulinum toxin refers to an isolated, or essentially orsubstantially isolated, botulinum toxin protein having a molecularweight of about 150 kD. That is, “non-complexed” forms exclude non-toxinproteins, such as non-toxin hemagglutinin and non-toxinnon-hemagglutinin proteins, normally associated with the complexed form.“Non-complexed” botulinum toxin is used interchangeably herein with“free” botulinum toxin. All the botulinum toxin serotypes made by nativeClostridium botulinum bacteria are synthesized by the bacteria asinactive single chain proteins which are then cleaved or nicked byproteases to become neuroactive. The protein comprises an about 100 kDheavy chain joined by a disulfide bond to an about 50 kD light chain.

Botulinum toxin complexes can be dissociated into toxin and non-toxinproteins by various means, including, for example, raising the pH toabout 7.0, treating the complex with red blood cells at a pH of about7.3, and/or subjecting the complex to a separation process, such ascolumn chromatography in a suitable buffer at a pH of about 7 to about8.

The instant invention encompasses systems and methods that enablepurification of a non-complexed botulinum toxin, without associatednon-toxin proteins conventionally believed necessary during thepurification process to maintain stability. In preferred embodiments,the methods and systems described herein facilitate purification of thefree botulinum toxin without loss of stability. By “stability” or“stable” is meant that the botulinum toxin protein molecule retains boththe about 100 kD heavy chain and the about 50 kD light chain, joined toeach other by a disulfide bond, and in a conformation that allows forbiological activity.

In some embodiments, a particular system within the scope of the presentinvention is operated in conjunction with a particular method within thescope of the present invention. A system within the scope of the presentinvention can comprise a plurality (preferably consecutive series) ofchromatography columns for use with a corresponding plurality(preferably consecutive series) of chromatography steps. Further, asystem within the scope of the instant invention may comprise aplurality (preferably a consecutive series) of non-chromatographydevices, such as filtration and/or centrifugation apparatus, for usewith a corresponding plurality (preferably consecutive series) ofnon-chromatography steps, e.g., as pre-chromatography steps.

In preferred embodiments, a process within the scope of the presentinvention comprises obtaining a sample comprising botulinum toxin from afermentation culture; subjecting it to a number of pre-chromatographypurifications; and then passing it through a plurality of chromatographycolumns to obtain a highly purified, highly potent non-complexedbotulinum toxin. Such a purified free botulinum toxin finds use in thepreparation of pharmaceutical compositions comprising the free botulinumtoxin as an active ingredient.

The overall steps for both pre-chromatography and chromatographyprocesses for some preferred embodiments of the instant invention areillustrated in FIG. 1A. For comparison, FIG. 1B shows a conventionalmethod for obtaining purified botulinum toxin complex. Briefly, FIG. 1Bdepicts a process involving depth filtration of a fermentation culture,followed by tangential flow filtration of the filtrate obtained (using300 kD ultramicrofiltration); followed by a clarifying centrifugationstep. The pellet (insoluble fraction) resulting from the centrifugationstep is then re-suspended in sodium chloride, and loaded onto ahydrophobic interaction or ion exchange column. The chromatographicpurification step is repeated at least three times to give a finaleluent containing the 900 kD botulinum toxin type A complex.

Fermentation and Acid Precipitation

As FIG. 1A illustrates, the non-complexed botulinum toxin is generallypurified from a fermentation culture. A “fermentation culture” as usedherein refers to a culture or medium comprising cells, and/or componentsthereof, that are synthesizing and/or have synthesized at least onebotulinum toxin. For example, Clostridial bacteria, such as Clostridiumbotulinum, may be cultured on agar plates in an environment conducive tobacterial growth, such as in a warm anaerobic atmosphere. The culturestep typically allows Clostridial colonies with desirable morphology andother characteristics to be obtained. Selected cultured Clostridialcolonies then can be fermented in a suitable medium as a fermentationculture. The cultured cells may include non-Clostridial species as thehost cells, such as E. coli or yeast cells, that are rendered capable ofbiosynthesizing a botulinum toxin by recombinant technology. Suitablefermentation culture conditions can depend on the host cells used andare generally known in the art.

In preferred embodiments, fermentation may be allowed to progress tocompletion, such that cells are mature and have biosynthesized abotulinum toxin. Growth of Clostridium botulinum cultures is usuallycomplete after about 24 to about 36 hours. After a certain additionalperiod of time, the bacteria typically lyse and release into the mediumthe synthesized botulinum toxin complex in a complexed form. Forexample, during a fermentation of about 60 to about 96 hours, mostClostridium botulinum cells undergo lysis and release botulinum toxintype A complex.

In some embodiments, the fermentation culture can comprise one or moreanimal products, such as animal proteins, used in conventionalfermentation culture procedures. For example, botulinum toxin can beproduced by anaerobic fermentation of Clostridium botulinum using amodified version of the well known Schantz process (see e.g. Schantz E.J., et al., Properties and use of botulinum toxin and other microbialneurotoxins in medicine, Microbiol Rev 1992 March; 56(1):80-99; SchantzE. J., et al., Preparation and characterization of botulinum toxin typeA for human treatment, chapter 3 in Jankovic J, ed. Neurological Diseaseand Therapy. Therapy with botulinum toxin (1994), New York, MarcelDekker; 1994, pages 41-49, and; Schantz E. J., et al., Use ofcrystalline type A botulinum toxin in medical research, in: Lewis G EJr, ed. Biomedical Aspects of Botulism (1981) New York, Academic Press,pages 143-50, each incorporated herein by reference). Both the Schantzand the modified Schantz process for obtaining a botulinum toxin makeuse of animal products, including animal-derived-Bacto-Cooked Meatmedium in the culture vial, and casein in the fermentation media.Additionally, the Schantz toxin purification makes use of DNase andRNase from bovine sources to hydrolyze nucleic acids present in thefermentation culture.

However, administration of a pharmaceutical containing an activeingredient that was purified using a process involving animal-derivedproducts can subject a patient to a potential risk of receiving variouspathogenic agents. For example, prions may be present in apharmaceutical composition comprising contaminating animal-derivedproducts, such as the prion responsible for Creutzfeldt-Jacob disease.As another example, there is a risk of transmitting a spongiformencephalopathy (TSE), such as a bovine spongiform encephalopathy (BSE)when animal products are used in the process of making a pharmaceuticalcomposition. The use of a botulinum toxin obtained via processes free ofanimal products, however, reduces such risks. Therefore, in somepreferred embodiments, the invention provides a process that is free ofanimal products, or essentially or substantially animal-product-free(APF). “Animal product free”, “essentially animal product free”, or“substantially animal product free” encompasses, respectively, “animalprotein free”, “essentially animal protein free”, or “substantiallyanimal protein free” and respectively means the absence, essentialabsence, or substantial absence, of products derived from animals,non-limiting examples of which include products derived from blood orpooled blood. “Animal” is used herein to refer to a mammal (such as ahuman), bird, reptile, amphibian, fish, insect, spider or other animalspecies, but excludes microorganisms, such as bacteria and yeasts.

An animal-product-free process (or a substantially or essentially animalproduct-free-process) refers to a process that is entirely,substantially, or essentially free of animal-derived products, reagentsand proteins, such as immunoglobulins, other blood products,by-products, or digests; meat products, meat by-products, meat digests;and milk or dairy products, by-products or digests. Accordingly, anexample of an animal-product free fermentation culture procedure is afermentation process, such as bacterial culturing, which excludes blood,meat, and dairy products, by-products, and digests. Ananimal-product-free fermentation process for obtaining a non-complexedbotulinum toxin reduces the possibility of contamination with viruses,prions or other undesirable agents, which can then accompany the toxinwhen administered to humans.

Animal-product-free fermentation procedures using Clostridium culturesare described, e.g., in U.S. Pat. Nos. 7,452,697 and 7,354,740, herebyincorporated by reference. For example, the growth media for productionof the botulinum toxin may comprise vegetable-based products, instead ofanimal-derived products, such as soy-based products and/or thedebittered seed of Lupinus campestris. Soy-based fermentation media foruse in an animal product free fermentation culture, for example, cancomprise a soy-based product, a source of carbon such as glucose, saltssuch as NaCl and KCl, phosphate-containing ingredients such as Na₂HPO₄and KH₂PO₄, divalent cations such as iron and magnesium, iron powder,amino acids such as L-cysteine and L-tyrosine, and the like. Preferably,the soy is hydrolyzed soy and the hydrolyzation has been conducted usingenzymes not derived from animals. Sources of hydrolyzed soy include butare not limited to Hy-Soy (Quest International), Soy peptone (Gibco)Bac-soytone (Difco), AMISOY (Quest), NZ soy (Quest), NZ soy BL4, NZ soyBL7, SE50M (DMV International Nutritionals, Fraser, N.Y.), and SE50MK(DMV).

As FIG. 1A illustrates, in certain embodiments a sample comprisingbotulinum toxin is obtained from a fermentation culture. For example,after a certain period of fermentation, in either animal product free ornon-animal product free media, botulinum toxin complex is released intothe medium and can be harvested by precipitation. For example, in someembodiments, as in the well-known Schantz process, the fermentationmedium comprising the botulinum toxin may be subjected to acidprecipitation to encourage the botulinum toxin complexes to associatewith cell debris and form an acid precipitate. In some particularlypreferred embodiments, about 3 M sulfuric acid solution may be added tothe fermentation culture to form the acid precipitate. Preferably, thepH is reduced to about 3 to about 4, more preferably to about 3.2 toabout 3.8, and even more preferably about 3.5. In some embodiments, theculture temperature is also reduced, e.g., to about below 25° C., 24°C., 23° C., 22° C., 21° C., or 20° C. These conditions further enhancethe association of botulinum toxin complexes with cell debris. The acidprecipitate formed will comprise bound botulinum toxin complexes and canbe used as the starting material in further purification steps, such asclarification steps; whereas the filtrate is discarded.

In contrast, the conventional process depicted in FIG. 1B does notinclude an acid precipitation step. That is, while the purificationprocedure also begins with a fermentation culture comprising a botulinumtoxin complex, the culture medium is subjected to depth filtration, andthe filtrate, rather than the cell debris, is used in subsequentpurification steps. In the FIG. 1B process, the cell debris isdiscarded, rather than the filtrate, whereas, as illustrated in FIG. 1A,the filtrate is discarded and the cell debris (acid precipitate) is usedfor further purification steps, e.g., in the pre-chromatographypurifications discussed below.

Pre-Chromatography Purifications

In some embodiments, the sample obtained from the fermentation medium issubjected to one or more pre-chromatography purifications.Pre-chromatography purifications can include at least one of tangentialflow filtration, nuclease digest, and clarifying centrifugation and/orfiltration. A non-limiting example of a process flow containingpre-chromatography purification contemplated by the invention isprovided in FIG. 1A. As noted above, in preferred embodiments, thepre-chromatography procedures are carried out on a precipitate (orinsoluble fraction) of a fermentation culture comprising the botulinumtoxin, rather than on the fermentation culture itself or on a filtratederived therefrom, as in the process illustrated in FIG. 1B. That is, inpreferred embodiments of the invention, pre-chromatography(clarification) steps start with the acid precipitate (insolublefraction).

In some embodiments, the sample (acid precipitate or insoluble fraction)comprising a botulinum toxin is subjected to tangential flow filtration.Tangential flow filtration is a process typically used to clarify,concentrate, and/or purify proteins. In contrast to normal flowfiltration, where fluid moves directly towards a filter membrane underapplied pressure, in tangential flow filtration, the fluid movestangentially along, or parallel to, the surface of the membrane. Appliedpressure serves to force a portion of the fluid through the filtermembrane, to the filtrate side, while particulates and macromoleculestoo large to pass through membrane pores are retained. Unlike normalflow filtration, however, the retained components do not build up at themembrane surface but are swept along by the tangentially flowing fluid.In certain preferred embodiments, tangential flow filtration is used toconcentrate the insoluble material (cell debris) with which thebotulinum complex is associated, while permitting filtrate to passthrough the membrane pores. (See, e.g., FIG. 1A.) Tangential flowfiltration parameters, such as pore size, feed flow, applied pressure,and the like, may be selected by those of skill in the art toconcentrate cell debris and to produce a more concentrated samplecomprising the botulinum toxin complex. In some particularly preferredembodiments, for example, tangential flow filtration with filters havinga pore size of about 0.1 μm may be used.

In some embodiments, the sample comprising a botulinum toxin issubjected to nuclease digestion. Nuclease digestion can facilitateremoval of nucleic acid components with which the Botulinum toxincomplexes tend to associate. In certain preferred embodiments, nucleasedigestion follows tangential flow filtration and is carried out on theconcentrated cell debris obtained therefrom. (See, e.g., FIG. 1A.) Forexample, the concentrated cell debris sample may have its pH adjusted toallow nuclease activity and may be incubated with one or more suitablenucleases, such as DNases and/or RNases that digest (hydrolyze) DNAand/or RNA, respectively. Depending on the nuclease enzyme used,suitable pH may be about 5 to about 7, preferably about 6. In someembodiments, benzamidine is used as a protease inhibitor to preventproteolysis of the toxin during nuclease digestion step. The nucleaseused may be derived from any suitable source, including animal sourcesand/or non-animal sources.

In more preferred embodiments, the nuclease is obtained from anon-animal source, to provide an animal-product-free nuclease and ananimal-product-free process. Accordingly, the instant inventionencompasses animal-product-free processes and systems (or substantiallyor essentially animal product free processes and systems) for purifyingbotulinum toxin which comprise use of a nuclease. An animal-product-freenuclease may be made recombinantly, e.g., using recombinant bacteria,yeasts, or other suitable microorganisms, which have been transformed toexpress a DNase and/or RNase for use in a nuclease digestion stepaccording to processes described herein. Nuclease digestion typicallyreduces the nucleic acid content of the sample, as the host cell nucleicacids are degraded and their removal is facilitated. For example,hydrolyzed nucleic acids and other low molecular weight impurities canbe removed by further purification steps.

In certain embodiments, the sample comprising a botulinum toxin may besubjected to clarifying centrifugation and/or filtration. Clarifyingcentrifugation or filtration refers to centrifugation or filtrationsteps used to remove gross elements, such as whole and lysed cells andcell debris, from the sample, resulting in a measurably clearer sample.In certain embodiments, the centrifugation is performed at about10,000×g to about 30,000×g, more preferably at about 15,000×g to about20,000×g, and most preferably at about 17,700×g. Clarifying filtrationwill typically comprise normal flow filtration, also called “dead end”filtration, where fluid is moved directly toward a filter media underapplied pressure, and particulates too large to pass through the filterpores accumulate at the surface or within the media itself, whilesmaller molecules pass through as the filtrate. In some particularlypreferred embodiments, the sample is mixed with ammonium sulfate andnormal flow filtration is performed using a filter with a pore size ofabout 0.1 to about 0.3 μm, and more preferably a pore size of about 0.2μm. (See, e.g., FIG. 1A.) In certain particularly preferred embodiments,one or more clarifying step(s) follow the nuclease digestion step. Incertain still more preferred embodiments, one or more clarifying step(s)immediately precede purification by chromatography.

Notably, in preferred embodiments, the clarified supernatant or filtrateprovides the botulinum toxin-containing sample for use in furtherpurification steps, such as the chromatography purification steps,rather than the insoluble fraction, which is discarded. This is incontrast with the process outlined in FIG. 1B, where the botulinum toxincomplex is contained in the insoluble fraction from pre-chromatographysteps that do not involve acid precipitation, such as e.g., as acentrifugation pellet, obtained from pre-chromatography centrifugation,and the supernatant is discarded.

Moreover, and again in contrast with the process outlined in FIG. 1B,the pre-chromatography steps in some embodiments of the invention do notrequire a tangential flow filtration step of a filtrate obtained fromfermentation culture. That is, the sample used for chromatographypurification in some embodiments of the invention is not obtained bysubjecting a soluble fraction of the fermentation culture to tangentialflow filtration. Rather, in certain embodiments, the present inventionuses insoluble material (such as an acid precipitate), eliminating anystep where a fermentation culture filtrate is subjected to tangentialflow filtration in an attempt to concentrate soluble botulinum toxincomplexes. Thus, in preferred embodiments, the pre-chromatography stepsof the invention eliminate the need for any such step, by instead usingacid to precipitate the desired toxin complexes with other insolublematerial (cell debris).

Chromatography Purification Steps

FIG. 1A also illustrates chromatographic purification steps according tocertain embodiments of the instant invention. According to oneembodiment of the invention, chromatographic methods for purifying anon-complexed botulinum toxin comprise passing a sample comprisingbotulinum toxin through a plurality of chromatography columns to obtaina highly purified, highly potent, non-complexed form of the neurotoxin.

In certain embodiments, a complexed botulinum toxin is separated fromother cellular components using a hydrophobic interaction column (seee.g., FIG. 1A). This column captures the botulinum toxin in complexedform, while allowing impurities to flow through the column. The columnused may be any hydrophobic interaction column known in the art suitablefor such purpose, such as Butyl Sepharose Fast Flow column or PhenylSepharose HP, commercially available from GE Healthcare Life Sciences.In some embodiments, the method further comprises conditioning thesample for hydrophobic interaction chromatography before loading ontothe column. For example, for use in the Phenyl Sepharose HP column, thesample may be combined with a 0.5M ammonium sulfate solution at pH 6,and 50 mM phosphate before loading. Other columns, buffers and pHconditions that may be used include columns such as Phenyl SepharoseFast Flow high substitution, Phenyl Sepharose Fast Flow lowsubstitution, Butyl Sepharose, and Octyl Sepharose; buffers such asacetate, citrate, MES, histidine, piperazine, and malonate, each in thepH range of about 4.0 to about 7.0, more preferably about 4.5 to about6.5, and even more preferably about 5.5. Other buffer and pH conditionsmay be determined to optimize yield from a particular column used, asknown in the art, based on the teachings provided herein. Withoutwishing to be bound to theory, it is believed that separation involvesbinding of the toxin complex to resin at a pH below 7, to avoiddissociation at this step, while allowing many cell-derived impuritiesto flow through, such as, e.g., smaller proteins, nucleic acids, and thelike.

For eluting the captured (bound) toxin from the hydrophobic interactioncolumn, a suitable buffer can be used, as known in the art. In someparticularly preferred embodiments, a descending gradient of ammoniumsulfate is used. The concentration range of the descending gradient maybe from about 0.6 M to about 0.0 M, about 0.5 M to about 0.0 M, or about0.4 M to about 0.0 M. Other eluting buffers that may be used include,for example descending gradients of sodium sulfate (Na₂SO₄); sodiumchloride (NaCl); potassium chloride (KCl); ammonium acetate (NH₄OAc);and the like. Fraction(s) containing a product peak can be identified,as known in the art. The peak fraction is typically found, e.g., whenusing ammonium sulfate, in a concentration range of about 0.4 M to about0.0 M; more preferably about 0.3 M to about 0.0 M; and most preferablyabout 0.25 M to about 0.0 M ammonium sulfate, while the pH is kept atabout 6 to maintain the complex. That is, the fraction(s) containing theeluted botulinum toxin complex can be identified and used in subsequentpurification steps.

In preferred embodiments, the botulinum toxin complex obtained is causedto dissociate to give a non-complexed form. In certain preferredembodiments, the dissociation step is performed after the hydrophobicinteraction chromatography step and/or before subsequent chromatographysteps (e.g., see FIG. 1A). Accordingly, in some preferred embodiments,the instant invention encompasses methods and systems where thechromatographic target molecule differs from one chromatographic step toanother. That is, in an initial chromatographic step, the targetcomprises a botulinum toxin complex, whereas in subsequentchromatographic steps, the target comprises the free botulinum toxin,dissociated from non-toxin proteins such as hemagglutinin andnon-hemagglutinin proteins. In contrast, the process outlined in FIG. 1Binvolves chromatography steps that are all designed to purify botulinumtoxin complexes.

Dissociation of the botulinum toxin complex to produce the non-complexedbotulinum toxin protein may be achieved in a number of ways, e.g., asknown in the art and/or described herein. For example, dissociation maybe achieved by raising the pH to about 7.0; or, in embodiments in whichanimal protein free purification is not necessary, treating the complexwith red blood cells at a pH of about 7.3.

In a preferred embodiment and to provide animal free toxin, the complexis subjected to a separation process based on adjustment pH of thecomplex in a suitable buffer Suitable buffers include, but are notlimited to, cationic buffers, preferably cationic buffers that will notinteract or will not substantially interact with the anion exchangecolumn. Suitable cationic buffers include, e.g., Tris, bis-Tris,triethanolamine, N-methyl diethanolamine. A pH of between about 7 toabout 8.4; more preferably between about 7.4 to about 8.2; and mostpreferably a pH of about 7.8 is typically suitable for dissociating thecomplex to release the non-complexed botulinum toxin. In someparticularly preferred embodiments, for example, the pH of the eluent ofthe hydrophobic interaction column is raised to about 7.5, about 7.8, orpreferably to about 8.0. For example, in some embodiments, the eluentmay be diluted into a Tris buffer having a pH of about 7.8 to cause thecomplex to dissociate into individual components, including the about150 kD non-complexed botulinum toxin protein. The resulting mixturecomprising dissociated components can then be subjected to one or moreadditional chromatography purification steps, such as ion exchangechromatography steps designed to capture and further purify thenon-complexed toxin.

In certain embodiments according to the invention, the non-complexedbotulinum toxin may be purified using one or more ion exchangechromatography steps, (e.g., see FIG. 1A). Ion exchange chromatographyachieves fractionation based on electrostatic charge. The extent towhich a given protein binds to the column matrix is a function of theprotein's net charge, based on its individual amino acid composition andthe charge of the column matrix. Cationic ion exchange columns have netpositive charged matrix whereas anionic ion exchange columns have a netnegative charged matrix. Bound proteins can be selectively eluted fromthe column using a solvent (the eluant) containing a charged substance,such as salt ions, which competes with the charged matrix support forbinding to the charged proteins. Bound proteins can be thus fractionatedon the basis of the strength of their charge. Alternatively, proteinsmay be eluted by adjusted the pH which may alter the net charge of theprotein thereby altering its affinity to the charged matrix.

According to some preferred embodiments of the invention, the mixturecomprising non-complexed botulinum toxin is loaded onto an anionexchange column (e.g., see FIG. 1A). Notably, this column captures thebotulinum toxin in non-complexed form, such that the toxin protein anddissociated non-toxin proteins can be eluted in separate fractions. Thecolumn used may be any anion column known in the art suitable forseparating charged proteins, non-limiting examples of which include QSepharose HP, Q Sepharose Fast Flow, or Q XL Sepharose, commerciallyavailable from GE Healthcare Life Sciences. In some particularlypreferred embodiments, a Q XL Sepharose column is used. In someembodiments, the method further comprises conditioning the mixturecomprising the non-complexed botulinum toxin for anion exchangechromatography before loading onto the column. For example, buffer andpH conditions may be determined to optimize yield from the particularcolumn used, as known in the art, based on the teachings providedherein. For loading and use in the column, e.g., suitable buffersinclude, but are not limited to, cationic buffers, preferably cationicbuffers that will not interact or will not substantially interact withthe anion exchange column. Suitable cationic buffers include, e.g.,Tris, bis-Tris, triethanolamine, N-methyl diethanolamine. For loadingand equibrilating the column, a pH of between about 7.2 to about 8.6;more preferably between about 7.4 to about 8.2; and most preferably a pHof about 7.8 may be used.

For eluting the captured (bound) toxin and other dissociated componentsfrom the anion exchange column, a suitable buffer can be used, as knownin the art. Examples of suitable buffers include, for example, sodiumchloride (NaCl); and potassium chloride (KCl). In some particularlypreferred embodiments, an ascending gradient of sodium chloride is used.For example, a sodium chloride buffer having a concentration range fromabout 0.0 M to about 0.4 M NaCl, more preferably from about 0.0 M toabout 0.5 M NaCl, and even more preferably about 0.0 M to about 0.6 MNaCl may be used. Impurities separated in different fractions mayinclude, e.g., one or more non-toxin proteins of the dissociatedcomplex, such as, the non-toxin hemagglutinin and/or non-toxinnon-hemagglutinin proteins. Fraction(s) containing a product peak can beidentified, as known in the art. The peak may occur, for example, atabout 8 mSem to about 22 mSem at a pH between about 7.4 to about 8.4,and preferably at about 7.8, corresponding to about 0.08 M to about 0.18M NaCl. Conversely, other impurities may elute at about 30 to about 45mSem, corresponding to about 0.25 M to about 0.35 M NaCl.

The fraction(s) containing the eluted non-complexed botulinum toxin canbe identified to provide an eluent comprising a non-complexed botulinumtoxin. The peak may be identified by methods as known in the art, e.g.,using HPLC, western blot analysis, ELISA, non-reduced SDS-PAGE, and thelike. SDS-PAGE under non-reducing conditions, for example, can identifythe about 150 kDa toxin band, whereas other impurities will appear atbands corresponding to smaller molecules. This eluent comprising anon-complexed form may then be subjected to further chromatographicpurification steps.

In one particularly preferred embodiment, toxin purity is assessed bySDS-PAGE. As the skilled artisan will appreciate, SDS-PAGE analysis canbe conducted in the absence or presence of agents that cleave disulfidebonds present in the protein (i.e., non-reducing or reducing conditions,respectively). For example, with respect to botulinum toxin type A, themature and active form of the botulinum toxin type A protein molecule iscomprised of two polypeptide chains of 100 kD and 50 kD, respectively,which are held together by non-covalent interactions as well as adisulfide bond. When botulinum toxin type A produced by the inventiveprocess is assayed using non-reducing conditions, the botulinum toxintype A protein molecules migrate as a single protein band ofapproximately 150 kD and the measured purity is typically greater than98%. When the botulinum toxin type A protein amount loaded per gel laneis held to be within the dynamic range of the densitometer, then thereare few, if any, detectable impurity bands resulting in a measuredpurity of 100%. When the type A botulinum toxin is overloaded such thatthe main toxin band is above the dynamic range of the densitometer, thensome minor impurity bands may be detectable (as much as 1-2%).

However, when the SDS-PAGE analysis of botulinum toxin type A isconducted under reducing conditions, then the disulfide bond of thebotulinum toxin is cleaved and the botulinum toxin type A proteinmigrates as two components having molecular weights of 100 kD and 50 kD,respectively. When the botulinum toxin type A protein is loaded suchthat the main species are above the dynamic range of the densitometerand the SDS-page is run under reducing conditions then minor impurityspecies can be more easily detected. For instance, under theseconditions there may be as much as 5% of the 150 kD species present dueto incomplete proteolytic processing during the fermentation andrecovery process. Under these conditions the inventive process yields atoxin product (comprised of the active, cleaved 100 kD and 50 kDpolypeptide chains) that is typically greater than 90% of total proteinand more likely greater than 95% of total protein. Thus, the reportedmeasured purity of the toxin depends on the details of the SDS-PAGEmethod employed, as described herein. Furthermore, while the foregoingexample concerns botulinum toxin type A, the skilled artisan willappreciate that the SDS-PAGE analysis described herein can be readilyadapted to assess the purity of other serotypes of botulinum toxin.

In certain embodiments, the eluent from the anionic column comprisingnon-complexed botulinum toxin is loaded onto a cation exchange column(see, e.g. FIG. 1A). Notably, this column also captures the botulinumtoxin in non-complexed form, such that the toxin protein and dissociatednon-toxin proteins can be eluted in separate fractions. The column usedmay be any cation column known in the art suitable for separatingproteins, non-limiting examples of which include an SP Sepharose column,including SP Sepharose HP or SP Sephrose Fast Flow; a Mono S column; ora Source-S column, such as a Source-30S column, or preferably aSource-15S column, both commercially available from GE Healthcare LifeSciences. In some embodiments, the method further comprises conditioningthe eluent from the anionic exchange columns comprising non-complexedbotulinum toxin for cation exchange chromatography before loading ontothe column. In some preferred embodiments, the pH is adjusted so thatthe pH of the eluent being loaded on the column allows for efficientbinding of the free toxin to the column. For example, the pH can bemaintained within a range of from about 4 to about 8, preferably fromabout 5 to about 7.5, more preferably from about 6 to about 7, and mostpreferably at about 7. Further, in some embodiments, the eluent from theanionic column can be treated to reduce conductivity before loading ontothe cation exchange column, e.g., using a sodium phosphate buffer, anon-limiting example of which is a sodium phosphate buffer of about 20mM NaH₂PO₄. For example, the eluent from the anionic column may containas much as about 0.15 M NaCl, so that diluting in an about 20 mM NaH₂PO₄buffer reduces conductivity. In some specific embodiments, conductivityis reduced from about 12 mSem to about 3.3 mSem. Dilution in buffer,dialysis or other methods known in the art also may be used to reducethe conductivity.

For loading and use in the column, e.g., suitable buffers include, butare not limited to, anionic buffers, preferably anionic buffers thatwill not interact or will not substantially interact with the cationicexchange column. Suitable anionic buffers include, e.g., as MES, HEPES,and the like, and preferably sodium phosphate buffer. For loading andequibrilating the column, a pH of between about 4 to about 8; preferablybetween about 5 to about 7.5; more preferably from about 6 to about 7;and most preferably a pH of about 6.8 to about 7 may be used.

For eluting the captured (bound) toxin from the cation exchange columnseparately from other dissociated non-toxin proteins and otherimpurities, a suitable buffer can be used, as known in the art. In someparticularly preferred embodiments, an ascending gradient of sodiumchloride is used. A suitable concentration range for the sodium chloridegradient may be from about 0.0 M to about 1 M NaCl. Other salts that maybe used include, e.g., potassium chloride, that may be used at aconcentration gradient of about 0.0 M to about 0.5 M KCl. Fraction(s)containing a product peak can be identified, as known in the art. Thepeak may occur, for example from about 18 to about 25 mSem,corresponding to about 0.3 M to about 0.4 M NaCl, at a pH of about 6.7.That is, the fraction(s) containing the eluted non-complexed botulinumtoxin can be identified to provide an eluent from the cationic columncomprising non-complexed botulinum toxin. In particularly preferredembodiments, the eluent from the cationic column represents anon-complexed botulinum toxin of high purity, in high yield and havinghigh activity. In contrast, the process outlined in FIG. 1B provides a900 kD botulinum toxin type A complex in the final eluent.

Purified Non-Complexed Botulinum Toxin Product

The methods and systems described herein are useful to provide anon-complexed botulinum toxin of high purity, in high yield, and havinghigh activity. See Example 1 below. The product is also readilystabilized and conveniently used for the preparation of safepharmaceutical compositions.

In some preferred embodiments, the purified non-complexed botulinumtoxin is at least about 80% pure, preferably at least about 90% pure,more preferably at least about 95% pure, even more preferably at leastabout 98% pure, and most preferably at least about 99% pure, or evenabout 100% pure. “Purified non-complexed botulinum toxin” refers to afree botulinum toxin protein molecule that is isolated, or substantiallyisolated, from other proteins and impurities, which can otherwiseaccompany the non-complexed botulinum toxin as it is obtained from aculture or fermentation process. A purified non-complexed botulinumtoxin that is, for example, “80% pure” refers to an isolated orsubstantially isolated non-complexed botulinum toxin protein wherein thetoxin protein comprises 80% of total protein present as determined by orother suitable analytical methodology, non-limiting examples of whichinclude SDS-PAGE, CE, and HPLC. For example, in some preferredembodiments, the cationic column eluent comprising the non-complexedbotulinum toxin is at least about 99% pure, and contains less than about1% of host cell proteins that are not the approximately 150 kD botulinumtoxin originally present.

In some preferred embodiments, the purified non-complexed botulinumtoxin has an activity of at least about 150 LD₅₀ units/ng, preferably atleast about 180 LD₅₀ units/ng, more preferably at least about 200 LD₅₀units/ng, even more preferably at least about 210 LD₅₀ units/ng, andmost preferably at least about 220 LD₅₀ units/ng. One unit of botulinumtoxin is defined as the LD₅₀ upon intraperitoneal injection into femaleSwiss Webster mice weighing about 18-20 grams each. In other words, oneunit of botulinum toxin is the amount of botulinum toxin that kills 50%of a group of female Swiss Webster mice. “Activity” is usedinterchangeably herein with related expressions “biological activity”,“potency” and “toxicity” to described the action of a botulinum toxin.

In preferred embodiments, the non-complexed botulinum toxins obtainableby processes and systems described herein demonstrate biologicalactivity. That is, in preferred embodiments, the biological activity ortoxicity of the product is not lost upon purification in accordance withpreferred embodiments of the present invention, even though non-toxinproteins natively associated with the toxin protein are removed duringpurification. In even more preferred embodiments, the potency obtainedusing a given set of processes and parameters within the scope of theinvention is consistent and/or reproducible. For example, the potencymeasurement can be made with less than about 40% variability, preferablyless than about 35% variability, more preferably less than about 30%variability, even more preferably less than about 25% variability, andmost preferably less than about 20% variability.

In some preferred embodiments, the purification process provides thenon-complexed botulinum toxin in high yield. For example, the yieldobtained from 30 L of a fermentation culture may be at least about 30mg, preferably at least about 40 mg, more preferably at least about 70mg, even more preferably at least about 80 mg, and most preferably atleast about 90 mg, corresponding to a yield of at least about 1 mg/L,preferably at least about 1.3 mg/L, more preferably at least about 2.3mg/L, even more preferably at least about 2.7 mg/L, and most preferablyat least about 3 mg/L, respectively. In even more preferred embodiments,the yield obtained using a given set of processes and parameters withinthe scope of the invention is reproducible. For example, yield can bemeasured with less than about 40% variability, preferably less thanabout 35% variability, more preferably less than about 30% variability,even more preferably less than about 25% variability, and mostpreferably less than about 20% variability.

In some particularly preferred embodiments, the purified non-complexedbotulinum toxin is stable during purification using the processes andsystems described herein. It has been believed that removal ofassociated non-toxin proteins from a botulinum toxin complex, such asbotulinum toxin type A complex, results in a markedly unstable botulinumtoxin product. The instant invention, however, provides methods andsystems that can stably isolate free botulinum toxin, without associatednon-toxin proteins conventionally believed necessary during thepurification process to maintain stability, as discussed above.

In some preferred embodiments, methods and systems described hereinprovide a non-complexed botulinum toxin that requires very fewpost-chromatography steps, e.g., in terms of maintaining stabilityduring storage, and in terms of applicability to pharmaceutical uses.For example, as known in the art, ammonium sulfate may be added to thefree botulinum toxin to prepare an ammonium sulfate suspension forstorage. The composition comprising free botulinum toxin and ammoniumsulfate may be readily stored in a refrigerator and later can be readilyretrieved for use in pharmaceutical applications. Indeed, the stability,high yield and purity, and high and consistent potency of the toxinobtainable by methods described herein facilitate pharmaceutical use ofthe purified product, as described in more detail below.

Uses of Purified Non-Complexed Botulinum Toxin

The non-complexed botulinum toxin purified according to this inventioncan be used in the preparation of pharmaceutical compositions comprisingthe toxin as an active ingredient for administration to any subject whowould receive a benefit from such pharmaceutical compositions. Inpreferred embodiments, the subjects to be treated are mammals,preferably humans. “Pharmaceutical composition” as used herein refers toa formulation in which an active ingredient can be a botulinum toxin.The formulation will contain at least one additional ingredient and besuitable for diagnostic, therapeutic, and/or or cosmetic administrationto a subject, such as a human patient. The pharmaceutical compositioncan be liquid or solid; and may be a single or multi-component system,for example a lyophilized composition reconstituted with a diluent suchas saline.

Another aspect of the invention provides for administration of apurified botulinum toxin molecule to a patient. “Administration” as usedherein refers to providing a pharmaceutical composition to a subject orpatient. The pharmaceutical composition may be administered by, anymethod known in the art, including e.g., intramuscular (i.m.),intradermal, intranasal, or subcutaneous administration, intrathecaladministration, intracranial, intraperitoneal (i.p.) administration, ortopical (transdermal) and implantation (e.g., of a slow-release device)routes of administration. In certain preferred embodiments, the purifiednon-complexed botulinum toxin is administered topically or by injectionin compositions as described in U.S. patent application Ser. Nos.09/910,432; 10/793,138; 11/072,026; 11/073,307, 11/824,393, and12/154,982, which are hereby incorporated by reference in theirentirety.

In certain embodiments, compositions comprising non-complexed botulinumtoxin in an ammonium sulfate suspension can be readily compounded into apharmaceutical composition. For example, an ammonium sulfate suspensioncomprising non-complexed botulinum toxin protein can be centrifuged torecover the protein and the protein can be re-solubilized, diluted, andcompounded with one or more pharmaceutically acceptable excipients. Incertain embodiments, the pharmaceutical composition may comprise anon-complexed botulinum toxin as an active pharmaceutical ingredient,and may further comprise one or more buffers, carriers, stabilizers,preservatives and/or bulking agents. The pharmaceutical compositions maybe lyophilized to powder for storage, and re-constituted for furtheruse. Accordingly, processes and systems described herein can provide abotulinum toxin in a form particularly suited to pharmaceuticalapplications terms of ease of preparation.

The pharmaceutical composition may find use in therapeutic, diagnostic,research and/or cosmetic applications. For example, as discussed above,botulinum toxin type A is clinically used to treat neuromusculardisorders characterized by skeletal muscle hyperactivity, such asessential blepharospasm, strabismus, cervical dystonia, and glabellarline (facial) wrinkles. Moreover, in certain applications, non-complexed(about 150 kD) botulinum toxin is the preferred form for treatinghumans. See, e.g., Kohl A., et al., Comparison of the effect ofbotulinum toxin A (Botox®) with the highly-purified neurotoxin (NT 201)in the extensor digitorum brevis muscle test, Mov Disord 2000; 15 (Suppl3):165. Accordingly, certain botulinum toxin pharmaceutical compositionsare preferably prepared using non-complexed botulinum toxin, as opposedto a botulinum toxin complex.

EXAMPLES Example 1 Comparison of Inventive Process with a ModifiedSchantz Process

Purifications of non-complexed botulinum toxin type A using processeswithin the scope of the instant invention (‘inventive process”) weredirectly compared to purifications based on the traditional Schantzapproach, further modified by the addition of chromatographic steps toprovide the non-complexed form (Modified Schantz process”). Briefly,Clostridium botulinum bacteria were cultured and allowed to grow untilfermentation was complete (usually about 72 to about 120 hours frominoculation to harvest). A volume of 30 L of the fermentation culturethen was used in each of the following purification procedures.

The modified Schantz process used involved typical acidification of thefermentation culture to precipitate the toxin, followed byultramicrofiltration (UF) and diafiltration (DF) to concentrate the rawtoxin. DNase and RNase were added to the harvested toxin to digest(hydrolyze) nucleic acids, which were then removed by an additional UFstep, using tangential flow filtration (300 kD UF). The toxin was nextextracted with phosphate buffer, followed by three sequentialprecipitation steps: cold ethanol precipitation; hydrochloric acidprecipitation, and ammonium sulfate precipitation, where thesupernatants each time were normally discarded. This procedure provideda 900 kD botulinum toxin type A complex, which was then subjected toadditional chromatography steps to provide the free toxin. Specifically,the toxin complex was resolubilized and subjected to negative batchadsorption onto a DEAE resin. The eluent was then run on a gravity flowanion exchange column (DEAE-Sepharose), followed by a gravity flowcation exchange column (CM-Sepharose). Yield was determined, the lengthof time the process took was recorded (not counting the fermentationperiod), and the purified non-complexed botulinum toxin type A wasmeasured for purity by SDS-PAGE analysis and assayed for potency, e.g.,by techniques known to those skilled in the art. The entire modifiedSchantz process was repeated for three different lots, lot numbers 1, 2and 3, and the results recorded in Table 1 below.

The inventive process was used with three different lots, lot numbers 4,5 and 6, in accordance with systems and methods described herein.Briefly, the fermentation culture was subjected to acid precipitationusing 3M sulfuric acid to reduce pH to 3.5, at a temperature below 25°C. The acid precipitate was then subjected to 0.1 μm tangential flowfiltration to concentrate cell mass. The pH then was adjusted to 6 andnucleases added to reduce host cell nucleic acid content, followed byclarification by centrifugation to remove cell debris and dead endfiltration at 0.2 μm with added ammonium sulfate. The filtrate was thendirectly loaded onto the hydrophobic interaction column, PhenylSepharose HP (GE Life Sciences), eluted with a descending gradient ofammonium sulfate, and the product peak isolated. The eluent was thendiluted into Tris buffer pH 7.8 to dissociate the toxin complex, whichthen was loaded onto the anion exchange column Q XL Sepharose (GELifesciences), eluted with an ascending gradient of sodium chloride, andagain the product peak collected. This eluent was then diluted in asodium phosphate buffer (to reduce conductivity) and loaded onto eitherthe anion exchange column, Q XL Sepharose (for lots #4 and 5), or thecation exchange column, Source-S (GE Life Sciences) (for lot #6), againeluted with an ascending gradient of sodium chloride, and a finalproduct peak collected and stored. This process yielded non-complexedbotulinum toxin type A. Yield was determined, the length of process timerecorded (not counting the fermentation period), and the toxin measuredfor purity by SDS-PAGE analysis and assayed for potency, e.g., bytechniques known to those skilled in the art. Results also recordedTable 1 below.

TABLE 1 Modified Schantz Process Inventive Process Lot # 1 2 3 4 5 6Process 10 days 4 days Time % Purity  99 n/a  97   98.6   95.3 100 Yield(30 L 11 mg 0 mg 4 mg 43 mg 99 mg 89 mg scale) Potency 255 n/a 173 259252 250 (LD50 Units/ng)

As Table 1 indicates, there was a lot failure with respect to lot #2 inthe modified Schantz process. The total lot failed giving zero yield.There was also a partial lot failure with respect to lot #3. There thefailure occurred at the hydrochloric acid precipitation step, but someproduct was rescued from the normally discarded supernatant. The rescuedproduct was reprocessed with a deviation step, accounting for theobserved reduced yield compared with lot #1 (4 mg compared with 11 mg)and the observed reduced potency compared with lot #1 (173 LD50 units/ngcompared with 255 LD50 units/ng).

With respect to the lots used with the inventive process, lot #4 showeda reduced yield, compared to lot #5 for example (43 mg compared with 99mg) due to a chromatography system failure, involving high salt wash ofa column. With the failure, there was premature elution of a portion ofthe toxin, resulting in the observed reduced yield, but also an observedhigher purity (98.6% purity compared with 95.3% purity).

Lot #6 represents the results of a highly preferred embodiment of theinstant inventive processes and systems, where a cation exchange columnwas used in the third chromatography step. As Table 1 indicates, thisembodiment resulted in improved purity compared with lot #5 for example(100% purity compared with 95.3% purity), while high yield (89 mgcompared with 99 mg) and high potency (250 LD50 units/ng compared with252 LD50 units/ng) were maintained.

As Table 1 also indicates, the total length of the purification can beshortened in preferred embodiments of the instant invention. Forexample, lot #6 was purified within only 4 days, compared to the 10 daysit took to purify non-complexed botulinum toxin using the modifiedSchantz method that involved three additional chromatography steps afterthe conventional Schantz method.

The results indicate that the processes and systems taught herein can beused to prepare high yields of a non-complexed botulinum toxin, at highpotency and purity, and suggests that methods and systems describedherein can find use in large-scale efficient purification of anon-complexed botulinum toxin suitable for use, e.g., as an activeingredient in pharmaceutical compositions.

All references including patent applications and publications citedherein are incorporated herein by reference in their entirety and forall purposes to the same extent as if each individual publication orpatent or patent application was specifically and individually indicatedto be incorporated by reference in its entirety for all purposes. Manymodifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. A method for purifying a non-complexed botulinum toxin, the methodcomprising: (i) providing a crude non-complexed botulinum toxin; (ii)loading the crude non-complexed botulinum toxin on an anion exchangecolumn so as to permit capture of the non-complexed botulinum toxin bythe anion exchange column; (iii) eluting the non-complexed botulinumtoxin from the anion exchange column to give an eluent comprising thenon-complexed botulinum toxin; (iv) loading a cation exchange columnwith the eluent from the anion exchange column so as to permit captureof the non-complexed botulinum toxin by the cation exchange column; and(v) eluting purified non-complexed botulinum toxin from the cationexchange column.
 2. The method according to claim 1, wherein the crudenon-complexed botulinum toxin is obtained by obtaining a samplecomprising botulinum toxin complex; loading a hydrophobic interactioncolumn with the sample so as to permit capture of the botulinum toxincomplex by the hydrophobic interaction column; eluting the botulinumtoxin complex from the hydrophobic interaction chromatography column;and dissociating the botulinum toxin complex to obtain a mixturecomprising the crude non-complexed botulinum toxin.
 3. The methodaccording to claim 2, wherein the sample is a supernatant or filtratecomprising the botulinum toxin complex.
 4. The method according to claim2, wherein the sample is obtained by: subjecting a fermentation culturecomprising the botulinum toxin to acid precipitation to obtain an acidprecipitate; and performing tangential flow filtration on theprecipitate to concentrate precipitate.
 5. The method according to claim2, wherein the sample is obtained by subjecting an insoluble fraction ofa fermentation culture to tangential flow filtration.
 6. The methodaccording to claim 2, wherein the sample is subjected to a nucleasedigestion before loading on the hydrophobic interaction column.
 7. Themethod according to claim 6, wherein the nuclease is derived from ananimal product free process.
 8. The method according to claim 1, whereinthe method is substantially animal product free.
 9. The method accordingto claim 1, wherein the purified non-complexed botulinum toxin comprisesat least one of botulinum toxin type A, B, C₁, D, F, F and G.
 10. Themethod according to claim 1, wherein the purified non-complexedbotulinum toxin comprises a botulinum toxin type A.
 11. The methodaccording to claim 1, wherein the purified non-complexed botulinum toxinis at least 95% pure.
 12. The method according to claim 1, wherein thepurified non-complexed botulinum toxin has an activity of at least 200LD₅₀ units/ng.
 13. The method according to claim 1, wherein the methodproduces a yield of at least about 2 mg/L fermentation culture.
 14. Themethod according to claim 1 wherein the anionic column is selected fromthe group consisting of a Q Sepharose HP, Q Sepharose Fast Flow, and QXL Sepharose column, and wherein the cationic column is selected fromthe group consisting of a SP Sepharose, SP Sepharose HP, SP SephroseFast Flow, Mono S, Source-S, Source-30S, and Source-15S column.
 15. Themethod according to claim 1 wherein a buffer for loading thenon-complexed botulinum toxin onto the anionic column is selected fromthe group consisting of Tris, bis-Tris, triethanolamine, and N-methyldiethanolamine.
 16. The method according to claim 15 wherein the bufferis used at a pH from 7.4 to 8.2.
 17. The method according to claim 1wherein a buffer for loading the non-complexed botulinum toxin onto thecationic column is selected from the group consisting of sodiumphosphate, MES, and HEPES.
 18. The method according to claim 17 whereinthe buffer is used at a pH from 6.0 to 7.0.
 19. The method according toclaim 1 wherein pH of the anionic column is from 7.4 to 8.2.
 20. Themethod according to claim 1 wherein pH of the cationic column is from6.0 to 7.0.
 21. The method according to claim 1 wherein a gradient foreluting the non-complexed botulinum toxin from the anionic column isselected from the group consisting of an ascending gradient of sodiumchloride and an ascending gradient of potassium chloride.
 22. The methodaccording to claim 21 wherein the gradient is used at a pH from 7.4 to8.4.
 23. The method according to claim 1 wherein a gradient for elutingthe non-complexed botulinum toxin from the cationic column is selectedfrom the group consisting of an ascending gradient of sodium chlorideand an ascending gradient of potassium chloride.
 24. The methodaccording to claim 23 wherein the gradient is used at a pH from 6.0 to7.0.